Within the nucleus, pre-mRNA molecules are found encapsulated within a complex that contains several proteins and RNA molecules which are critical to the proper processing of mRNA and to the functions of the nuclear RNA processing machinery. These complexes, collectively known as heterogeneous nuclear ribonucleoproteins (hnRNPs) contain a unique set of six core proteins designated A1/A2, B1/B2 and C1/C2.
The first member of this subgroup, hnRNP A1 (also known as heterogeneous nuclear ribonucleoprotein core protein A1 and p40CRS), is believed to function in the stabilization, transport and processing (including alternative splicing) of newly synthesized mRNAs (Mayeda et al., Mol. Cell. Biol., 1993, 13, 2993-3001; Mayeda and Krainer, Cell, 1992, 68, 365-375; Munroe and Dong, Proc. Natl. Acad. Sci. U.S.A., 1992, 89, 895-899). The hnRNP A1 protein has also been classified as an apoptosis-associated protein and has been found to be specifically cleaved into three distinct fragments in a human Burkitt lymphoma cell line during antibody-induced apoptosis (Brockstedt et al., J. Biol. Chem., 1998, 273, 28057-28064). hnRNP A1 is a two-domain protein with the N-terminal (also referred to as UP1) harboring the single-stranded DNA and RNA binding properties, while the C-terminus contributes to the cooperative binding nature of the protein and contains both a nuclear localization and nuclear export signal (Casas-Finet et al., J. Mol. Biol., 1993, 229, 873-889; Kumar et al., J. Biol. Chem., 1990, 265, 17094-17100; Nadler et al., Biochemistry, 1991, 30, 2968-2976).
The protein shuttles continuously from the cytoplasm to the nucleus acting as a carrier protein for mRNAs (Cobianchi et al., Genetica, 1994, 94, 101-114). Compositions useful for the transport of molecules into the cell nucleus comprising appending a molecule to at least a portion of an hnRNP molecule are reported in the PCT publication Wo 93/17102. Also disclosed are methods to modulate the expression of an hnRNP protein by introducing into a cell oligonucleotides hybridizable to DNA coding for a hnRNP protein (Dreyfuss and Pinol-Roma, 1993). However, no oligonucleotide sequences targted to any of the greater than 20 hnRNP proteins are disclosed.
hnRNP A1 has also been shown to facilitate annealing of single-stranded nucleic acid molecules (Munroe and Dong, Proc. Natl. Acad. Sci. U.S.A., 1992, 89, 895-899) and to modulate the binding of snRNPs to intron sequences. Disclosed in U.S. Pat. No. 5,015,569 are methods directed at improving nucleic acid hybridization technology comprising the use of a sufficient amount of hnRNP A1 protein to accelerate the rate of hybridization of two complementary nucleic acid sequences in an in vitro assay (Pontius, 1991).
Another role described for hnRNP A1 includes participation in telomere biogenesis. Telomeres are comprised of long arrays of TTAGGG repeats found on the ends of mammalian chromosomes and that have, associated with them, specific DNA binding proteins. These structures function to preserve the integrity of chromosomes during the cell cycle by allowing the proper segregation during cell division. Mouse cell lines deficient in hnRNP A1 exhibit shorter telomeres than wild type counterparts. Restoring hnRNP A1 expression was shown to increase telomere length. These effects were further characterized to lie in the N-terminal domain of hnRNP A1 which is also referred to as UP1 (LaBranche et al., Nat. Genet., 1998, 19, 199-202). Disclosed in PCT publication WO 98/00537 is a method to modulate telomere length by administering to a cell an effective amount of nucleic acid sequence encoding hnRNP A1 or a fragment thereof. Also disclosed is a method of triggering cellular senescence comprising administering at least one of a ligand, specific to hnRNP and UP1, which interferes with the activity of hnRNP A1 or UP1 by increasing the length of the telomeres, where the ligand may be an antibody, antisense molecule or ribozyme. However, no specific antisense sequences are disclosed (Chabot, 1998).
hnRNP A1 has been implicated, via its ability to control splicing events, in the process of oncogenesis. It has been demonstrated in mice that retroviral insertion of the Friend murine leukemia virus occurs adjacent to the hnRNP A1 locus. This insertion eliminates hnRNP A1 expression and, because of the role of hnRNP A1 in donor splice site selection, implicates hnRNP A1 in the oncogenesis process (Ben-David et al., Mol. Cell. Biol., 1992, 12, 4449-4455). In human retroviral infections, hnRNP A1 has been shown to interact with regulatory elements in the 5' long terminal repeat of the human T-cell leukemia virus (HTLV), the causative agent in AIDS (Black et al., J. Virol., 1995, 69, 6852-6858).
The pharmacological modulation of hnRNP A1 activity, expression or function may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of hnRNP A1, consequently there remains a long felt need for agents capable of effectively inhibiting hnRNP A1 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of hnRNP A1 expression.
The present invention provides compositions and methods for modulating hnRNP A1 expression, including modulation of the truncated form of hnRNP A1, UP1.